Pseudomonas aeruginosa glutathione biosynthesis genes play multiple roles in stress protection, bacterial virulence and biofilm formation

Wongsaroj, Lampet; Saninjuk, Kritsakorn; Romsang, Adisak; Duang-nkern, Jintana; Trinachartvanit, Wachareeporn; Vattanaviboon, Paiboon; Mongkolsuk, Skorn

doi:10.1371/journal.pone.0205815

Cited by 49 publications

(41 citation statements)

References 60 publications

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“…RNA-seq analysis further corroborated that a number of genes, including ppyR, migA, PilG - pilK, chpA-chpD , associated with biofilm and motilities, were down regulated in Δ gshA Δ gshB (Attila et al, 2008; Lau et al, 2009). These phenotypes are consistent with a recently published study (Wongsaroj et al, 2018). Finally, in the course of our studies, we have also discovered that the expression of a subset of known T6SS genes are decreased in Δ gshA Δ gshB , suggesting that GSH also modulates the expression of T6SS system.…”

Section: Discussionsupporting

confidence: 94%

Glutathione Activates Type III Secretion System Through Vfr in Pseudomonas aeruginosa

Zhang

et al. 2019

Front. Cell. Infect. Microbiol.

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Glutathione (GSH) is the most abundant antioxidant in all living organisms. Previously, we have shown that a deletion mutant in the glutathione synthetase gene (Δ gshB ) decreases the expression of type III secretion system (T3SS) genes of Pseudomonas aeruginosa . However, the mechanism remains elusive. In this study, a comprehensive transcriptomic analysis of the GSH-deficient mutant Δ gshA Δ gshB was used to elucidate the role of GSH in the pathogenesis of P. aeruginosa . The data show that the expression of genes in T3SS, type VI secretion system (T6SS) and some regulatory genes were impaired. Δ gshA Δ gshB was attenuated in a mouse model of acute pneumonia, swimming and swarming motilities, and biofilm formation. Under T3SS inducing conditions, GSH enhanced the expression of T3SS in both wild-type PAO1 and Δ gshA Δ gshB , but not in Δ vfr . Genetic complementation of Δ vfr restored the ability of GSH to induce the expression of T3SS genes. Site-directed mutagenesis based substitution of cysteine residues with alanine in Vfr protein abolished the induction of T3SS genes by GSH, confirming that GSH regulates T3SS genes through Vfr. Exposure to H 2 O 2 decreased free thiol content on Vfr, indicating that the protein was sensitive to redox modification. Importantly, GSH restored the oxidized Vfr to reduced state. Collectively, these results suggest that GSH serves as an intracellular redox signal sensed by Vfr to upregulate T3SS expression in P. aeruginos a. Our work provides new insights into the role of GSH in P. aeruginosa pathogenesis.

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Section: Discussionsupporting

confidence: 94%

Glutathione Activates Type III Secretion System Through Vfr in Pseudomonas aeruginosa

Zhang

et al. 2019

Front. Cell. Infect. Microbiol.

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“…Our findings provide genetic evidence in support of a role for glutathione in the control of MexT in vivo. Although our findings indicate that GshA is essential for the activity of MexT, it remains to be determined whether the effects of GshA on biofilm formation, stress resistance, motility, and virulence in P. aeruginosa (54,55) are tied to its effects on the activity of MexT.…”

Section: Discussionmentioning

confidence: 84%

Loss of RNA Chaperone Hfq Unveils a Toxic Pathway in Pseudomonas aeruginosa

et al. 2019

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Hfq is an RNA chaperone that serves as a master regulator of bacterial physiology. Here we show that in the opportunistic pathogen Pseudomonas aeruginosa, the loss of Hfq can result in a dramatic reduction in growth in a manner that is dependent upon MexT, a transcription regulator that governs antibiotic resistance in this organism. Using a combination of chromatin immunoprecipitation with high-throughput sequencing and transposon insertion sequencing, we identify the MexT-activated genes responsible for mediating the growth defect of hfq mutant cells. These include a newly identified MexT-controlled gene that we call hilR. We demonstrate that hilR encodes a small protein that is acutely toxic to wild-type cells when produced ectopically. Furthermore, we show that hilR expression is negatively regulated by Hfq, offering a possible explanation for the growth defect of hfq mutant cells. Finally, we present evidence that the expression of MexT-activated genes is dependent upon GshA, an enzyme involved in the synthesis of glutathione. Our findings suggest that Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of specific MexT-regulated genes. Moreover, our results identify glutathione to be a factor important for the in vivo activity of MexT. IMPORTANCE Here we show that the conserved RNA chaperone Hfq is important for the growth of the opportunistic pathogen Pseudomonas aeruginosa. We found that the growth defect of hfq mutant cells is dependent upon the expression of genes that are under the control of the transcription regulator MexT. These include a gene that we refer to as hilR, which we show is negatively regulated by Hfq and encodes a small protein that can be toxic when ectopically produced in wild-type cells. Thus, Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of MexT-regulated genes, including one encoding a previously unrecognized small protein. We also show that MexT activity depends on an enzyme that synthesizes glutathione.

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“…A Salmonella gshA deletion is hypersensitive to H 2 O 2 , and gshA and gshB deletions are hypersensitive to both nitric oxide (NO) and S-nitrosoglutathione (GSNO) 19 . Similarly, in Pseudomonas aeruginosa, gshA and/or gshB deletions are more sensitive to environmental stress and attenuated for virulence 20 . In Streptococcus pneumoniae, mutating gshT, an ABC transporter required for GSH import, increased S. pneumoniae sensitivity to superoxide, and was attenuated in a mouse model of infection 21 .…”

Section: Resultsmentioning

confidence: 99%

An intra-bacterial activity for a T3SS effector

Qaidi

Scott

Hays

et al. 2020

Sci Rep

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Many Gram-negative bacterial pathogens interact with mammalian cells by using type iii secretion systems (T3SS) to inject virulence proteins into host cells. A subset of these injected protein 'effectors' are enzymes that inhibit the function of host proteins by catalyzing the addition of unusual posttranslational modifications. The E. coli and Citrobacter rodentium NleB effectors, as well as the Salmonella enterica SseK effectors are glycosyltransferases that modify host protein substrates with N-acetyl glucosamine (GlcNAc) on arginine residues. This post-translational modification disrupts the normal functioning of host immune response proteins. T3SS effectors are thought to be inactive within the bacterium and fold into their active conformations after they are injected, due to the activity of chaperones that keep the effectors in a structural state permissive for secretion. While performing mass spectrometry experiments to identify glycosylation substrates of nleB orthologs, we unexpectedly observed that the bacterial glutathione synthetase (GshB) is glycosylated by NleB on arginine residue R256. NleB-mediated glycosylation of GshB resulted in enhanced GshB activity, leading to an increase in glutathione production, and promoted C. rodentium survival in oxidative stress conditions. these data represent, to our knowledge, the first intra-bacterial activity for a T3SS effector and show that arginine-GlcnAcylation, once thought to be restricted to host cell compartments, also plays an important role in regulating bacterial physiology.

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Pseudomonas aeruginosa glutathione biosynthesis genes play multiple roles in stress protection, bacterial virulence and biofilm formation

Cited by 49 publications

References 60 publications

Glutathione Activates Type III Secretion System Through Vfr in Pseudomonas aeruginosa

Glutathione Activates Type III Secretion System Through Vfr in Pseudomonas aeruginosa

Loss of RNA Chaperone Hfq Unveils a Toxic Pathway in Pseudomonas aeruginosa

An intra-bacterial activity for a T3SS effector

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